Patient-derived models: Advanced tools for precision medicine in neuroblastoma

Neuroblastoma is a childhood cancer derived from the sympathetic nervous system. High-risk neuroblastoma patients have a poor overall survival and account for ~15% of childhood cancer deaths. There is thus a need for clinically relevant and authentic models of neuroblastoma that closely resemble the human disease to further interrogate underlying mechanisms and to develop novel therapeutic strategies. Here we review recent developments in patient-derived neuroblastoma xenograft models and in vitro cultures. These models can be used to decipher mechanisms of metastasis and treatment resistance, for drug screening, and preclinical drug testing. Patient-derived neuroblastoma models may also provide useful information about clonal evolution, phenotypic plasticity, and cell states in relation to neuroblastoma progression. We summarize current opportunities for, but also barriers to, future model development and application. Integration of patient-derived models with patient data holds promise for the development of precision medicine treatment strategies for children with high-risk neuroblastoma

Inhibition of fatty acid synthesis induces differentiation and reduces tumor burden in childhood neuroblastoma

Many metabolic pathways, including lipid metabolism, are rewired in tumors tosupport energy and biomass production and to allow adaptation to stressful en-vironments. Neuroblastoma is the second deadliest solid tumor in children. Ge-netic aberrations, as the amplification of theMYCN-oncogene, correlate stronglywith disease progression. Yet, there are only a few molecular targets successfullyexploited in the clinic. Here we show that inhibition of fatty acid synthesis led toincreased neural differentiation and reduced tumor burden in neuroblastomaxenograft experiments independently ofMYCN-status. This was accompaniedby reduced levels of the MYCN or c-MYC oncoproteins and activation of ERKsignaling. Importantly, the expression levels of genes involved inde novofattyacid synthesis showed prognostic value for neuroblastoma patients. Our findingsdemonstrate that inhibition ofde novofatty acid synthesis is a promising pharma-cological intervention strategy for the treatment of neuroblastoma indepen-dently ofMYCN-status

Laser capture microdissection (LCM) and whole genome amplification (WGA) of DNA from normal breast tissue --- optimization for genome wide array analyses

<p>Abstract</p> <p>Background</p> <p>Laser capture microdissection (LCM) can be applied to tissues where cells of interest are distinguishable from surrounding cell populations. Here, we have optimized LCM for fresh frozen normal breast tissue where large amounts of fat can cause problems during microdissection. Since the amount of DNA needed for genome wide analyses, such as single nucleotide polymorphism (SNP) arrays, is often greater than what can be obtained from the dissected tissue, we have compared three different whole genome amplification (WGA) kits for amplification of DNA from LCM material. In addition, the genome wide profiling methods commonly used today require extremely high DNA quality compared to PCR based techniques and DNA quality is thus critical for successful downstream analyses.</p> <p>Findings</p> <p>We found that by using FrameSlides without glass backing for LCM and treating the slides with acetone after staining, the problems caused by excessive fat could be significantly decreased. The amount of DNA obtained after extraction from LCM tissue was not sufficient for direct SNP array analysis in our material. However, the two WGA kits based on Phi29 polymerase technology (Repli-g^®(Qiagen) and GenomiPhi (GE Healthcare)) gave relatively long amplification products, and amplified DNA from Repli-g^®gave call rates in the subsequent SNP analysis close to those from non-amplified DNA. Furthermore, the quality of the input DNA for WGA was found to be essential for successful SNP array results and initial DNA fragmentation problems could be reduced by switching from a regular halogen lamp to a VIS-LED lamp during LCM.</p> <p>Conclusions</p> <p>LCM must be optimized to work satisfactorily in difficult tissues. We describe a work flow for fresh frozen normal breast tissue where fat is inclined to cause problems if sample treatment is not adapted to this tissue. We also show that the Phi29-based Repli-g^®WGA kit (Qiagen) is a feasible approach to amplify DNA of high quality prior to genome wide analyses such as SNP profiling.</p

Mating system evolution and self-incompatibility in the wild plant species Brassica cretica

Compared to animals like ourselves, plants have a very flexible sexual life. Most plants are, for example, hermaphrodites with the potential capacity for reproduction by self-fertilization (or selfing). While selfing can provide several definite advantages for the individual plant, there is a downside; mainly the severe reduction in fitness due to inbreeding depression. To avoid the negative consequences of selfing, many hermaphrodite plant species have evolved an intricate self-recognition – or self-incompatibility (SI) – system that prevents fertilization by cognate pollen. SI is in the majority of cases genetically controlled by a narrowly delimited region of the genome, called the S locus. The S locus contains several tightly linked genes, two of which – SRK and SCR – determine the pistil (female) and pollen (male) SI recognition type. One of the best-characterized SI systems is found in the Brassicaceae family, which includes the model plant Arabidopsis thaliana and a number of economically important crop species of the Brassica genus, e.g. rape seed, cabbage, and turnip. For evolutionary biologists, SI have long been a prominent and fascinating example of Darwinian natural selection acting in a frequency-dependent manner, i.e. the rarer a genetic variant becomes, the more favoured by natural selection it is. For the S locus, this means that a very large number of variants – or haplotypes – are expected to be maintained in a population and that the DNA sequences of different haplotypes will be very divergent. However, until recently there has been a shortage of empirical studies from natural plant populations to test these, and other, theoretical predictions of S locus evolutionary dynamics. In this thesis, I have produced the largest SRK and SCR DNA sequence data set from a wild Brassica species available to date. These data have allowed me to explore, in more detail than previously possible, the population genetic properties and the evolutionary history of the Brassica S locus. Moreover, accompanying studies of the pattern of inheritance of S locus variants and the occurrence of self-fertilization in natural B. cretica population have added novel information of great value to the understanding of how plants produce offspring in nature

A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation

Amplification of the HER-2/neu (HER-2) proto-oncogene occurs in 10%–15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the HER-2 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine HER-2 amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples

Unequal segregation of SRK alleles at the S locus in Brassica cretica.

In the Brassicaceae plant family, which includes the Arabidopsis and Brassica genera, self-incompatibility (SI) is controlled by genes at the S locus. Using experimental crosses, we studied the pattern of inheritance of S-locus alleles in the wild species Brassica cretica. Four full-sib families were established and unequal segregation of alleles at the SRK SI gene was found in one family. The segregation distortion acted in favour of a recessive (class II) allele and was best explained by some form of gametic-level selection. Our findings are discussed in the light of theoretical predictions of differential accumulation of deleterious mutations among S-locus alleles

Nuclear and chloroplast microsatellites reveal extreme population differentiation and limited gene flow in the Aegean endemic Brassica cretica (Brassicaceae)

Nuclear and chloroplast microsatellite markers were used to study population structure and gene flow among seven Cretan populations of the Aegean endemic plant species Brassica cretica (Brassicaceae). Both nuclear and chloroplast markers revealed exceptionally high levels of population differentiation (overall FST = 0.628 and 1.000, respectively) and relatively little within-population diversity (overall HS = 0.211 and 0.000, respectively). Maximum-likelihood estimates of directional migration rates were low among all pairs of populations (average Nm = 0.286). There was no evidence that differences in flower colour between populations had any influence on historical levels of gene flow. In addition, a haplotype network showed that all five chloroplast haplotypes found in the sample were closely related. Together, these results suggest that current patterns of diversification in B. cretica are mainly a result of genetic drift during the last half million years. The main conclusions from the present study are consistent with the prevailing hypothesis that plant diversification in the Aegean region is driven by random rather than adaptive differentiation among isolated populations

A novel method for downstream characterization of breast cancer circulating tumor cells following CellSearch isolation.

Enumeration of circulating tumor cells (CTCs) obtained from minimally invasive blood samples has been well established as a valuable monitoring tool in metastatic and early breast cancer, as well as in several other cancer types. The gold standard technology for detecting CTCs in blood against a backdrop of millions of leukocytes is the FDA-approved CellSearch system (Janssen Diagnostics), which relies on EpCAM-based immunomagnetic separation. Secondary characterization of these cells could enable treatment selection based on specific targets in these cells, as well as providing a real time window into the metastatic process and offering unique insights into tumor heterogeneity. The objective of this study was to develop a method for downstream characterization of CTCs following isolation with the CellSearch system